As the field of Genomics has developed there has been an increasing need to isolate and analyze longer and longer contiguous stretches of nucleic acid. Methods for disease gene discovery and analysis would be facilitated by the ability to easily and rapidly obtain these sequences. For example, the analysis of genetic haplotypes, which require determining marker alleles over long contiguous DNAs, is currently very difficult and frequently done by statistical methods. A method that can simply and rapidly isolate long contiguous stretches of DNA would greatly simplify this analysis and enable new studies in this field.
Current methods for amplifying very long DNAs suffer from several drawbacks. Cheng et al., Proc. Natl. Acad. Sci. USA. 91(12): 5695-5699 (1994). The reactions are slow, with extension times on the order of 1.0 min per kb of extended amplicon. Thus a 20 kb amplicon takes more than 20 min of extension time per cycle or over 6 hours of PCR reaction time. Amplification of larger DNAs is prohibitively slow. Such extended incubations at the elevated temperatures of PCR tax the physical and chemical stability of the nucleotides, DNA and polymerase in the reactions and therefore make improvements difficult. Also the current commercial kit formulations for amplifying DNAs of this length require a mixture of two distinct DNA polymerases, a polymerase with a 3′-5′ proofreading activity and a non-proofreading DNA polymerase. Because this formulation requires two polymerases, it is difficult to optimize, troubleshoot or control. Thus, it is desirable to have a method for the rapid amplification of long nucleic acid fragments, using a simple, single enzyme formulation.
A thermostable DNA polymerase gene for Tba DNA polymerase was isolated and cloned from Thermococcus barossii, a thermophilic organism obtained from deep vent flange, Endeavor Segment, Juan de Fuca Ridge, off the coast of Washington State in the U.S.A. (Duffaud G D, Syst Appl Microbiol. 21(1):40-49 (1998)). Characterization of the purified Tba DNA polymerase showed that it possesses an active proofreading function in addition to its DNA-dependent DNA polymerase activity. Comparison to other DNA polymerases revealed that Tba DNA polymerase is a member of the Family B DNA polymerases and is approximately 80% conserved compared with the Pfu and T. littoralis DNA polymerases (U.S. Pat. No. 5,602,011).
Alteration of the FDIET sequence to FAIAT has been shown in some polymerases to decrease the nuclease activities. (Derbyshire, V. et al., Science 240:199-201, 1988; Bernad, A. et al., Cell 59:219-228, 1989; Frey, M. W. et al., Proc. Natl. Acad. Sci. USA 90:2579-2583, 1993; U.S. Pat. No. 5,489,523) U.S. Pat. No. 5,882,904 discloses that the Tba DNA polymerase can be engineered to have a reduced 3′-5′ exonuclease activity. Specifically, the FDIET amino acid sequence (residues 140-144 of native Tba DNA polymerase) has been altered to FAIAT to generated the exo* DNA polymerase.